1. Field of the Invention
This invention is in the field of biology and more particularly in the field of cell biology.
2. Description of the Prior Art
The ability to grow mammalian cells is important at both the laboratory and industrial levels. At the laboratory level, the limiting factor for cellular or viral research at the sub-cellular level is often the amount of raw material available to be studied. At the industrial level, there is much effort being devoted to the development of pharmaceuticals based on mammalian cell products. These are primarily vaccines for human viruses, but also include human growth hormone and other body hormones for medical applications.
Some mammalian cell types have been adapted for growth in suspension cultures. Examples of such cell types include HeLa (human), BHK (baby hamster kidney) and L mouse cells. Such cells, in general, have non-normal genetic complements, i.e., too many or too few chromosomes or abnormal chromosomes. Often, these cells will produce a tumor upon injection into an animal of the appropriate species.
Other mammalian cell types have not been adapted for growth in suspension culture to date, and will grow only if they can become attached to an appropriate surface. Such cell types are generally termed "anchorage-dependent" and include 3 T3 mouse fibroblasts, mouse bone marrow epithelial cells; Murine luekemia virus producing strains of mouse fibroblasts, primary and secondary chick fibroblasts; WI-38 human fibroblast cells; and, normal human embryo lung fibroblast cells (HEL299, ATCC CCL137 ). Some anchorage-dependent cells have been grown which are tumor causing but others were grown and found to be non-tumor causing. Also, some anchorage-dependent cells, such as WI-38 and HEL299, can be grown which are genetically normal.
Whereas considerable progress has been made in large scale mammalian cell propagation using cell lines capable of growth in suspension culture, progress has been very limited for large scale propagation of anchorage-dependent mammalian cells. Previous operational techniques employed for large scale propagation of anchorage-dependent cells were based on linear expansion. Cell culture plants utilized a large number of low yield batch reactors, in the forms of dishes, prescription bottles, roller tubes and roller bottles. Each of these was a discrete unit or isolated batch reactor requiring individual environmental controls. These controls, however, were of the most primative type due to economic considerations. Variation in nutrients was corrected by a medium change, an operation requiring two steps, i.e., medium removal and medium addition. Since it was not uncommon for a moderately sized facility to operate hundreds of these batch reactors at a time, even a single change of medium required hundreds of operations, all of which had to be performed accurately, and under exacting sterile conditions. Any multiple step operation, such as cell transfer or harvest, compounded the problem accordingly. Thus, costs of equipment, space and manpower were great for this type of facility.
There are alternative methods to linear scale-up from small batch cultures which have been proposed. Among such alternatives which have been reported in the literature are plastic bags, stacked plates, spiral films, glass bead propagators, artificial capillaries, and microcarriers. Among these, microcarrier systems offer certain outstanding and unique advantages. For example, great increases in the attainable ratio of growth surface to vessel volume (S/V) can be obtained using microcarriers over both traditional and newly developed alternative techniques. The increase in S/V attainable allows the construction of a single-unit homogeneous or quasi-homogeneous batch or semi-batch propagator for high volumetric productivity. Thus, a single stirred tank vessel with simple feedback control for pH and pO2 presents a homogeneous environment for a large number of cells thereby eliminating the necessity for expensive and space consuming, controlled environment incubators. Also, the total number of operations required per unit of cells produced is drastically reduced. In summary, microcarriers seem to offer economies of capital, space and manpower in the production of anchorage-dependent cells, relative to current production methods.
Microcarriers also offer the advantage of environmental continuity since the cells are grown in one controlled environment. Thus, microcarriers provide the potential for growing anchorage-dependent mammalian cells under one set of environmental conditions which can be regulated to provide constant, optimal cell growth.
One of the more promising microcarrier systems to date has been reported by van Wezel and involves the use of polydextran beads reacted with diethylaminoethyl (DEAE) in a stirred tank. A. L. van Wezel, "Growth of Cell Strains and Primary Cells on Microcarriers in Homogeneous Culture", Nature 216:64 (1967 ); D. van Hemert, D. G. Kilburn and A. L. van Wezel, "Homogeneous Cultivation of Animal Cells for the Production of Virus and Virus Products", Biotechnol. Bioeng. 11:875 (1969 ); and A. L. van Wezel, "Microcarrier Cultures of Animal Cells", Tissue Culture, Methods and Applications, P. F. Kruse and M. K. Patterson, eds., Academic Press, New York, p. 372 (1973 ). These beads are commercially produced by Pharmacia Fine Chemicals, Inc., Piscataway, New Jersey, under the tradename DEAE-Sephadex A50, an ion exchange system. Chemically, these beads are formed from a crosslinked dextran matrix having diethylaminoethyl groups covalently bound to the dextran chains. As commercially available, DEAE-Sephadex A50 beads have a particle size of 40- 120.mu. and a charge capacity of 3.5 .+-. 0.5 meq/gm dry material. Other anion exchange resins, such as DEAE-Sephadex A25, QAE-Sephadex A50 and QAE-Sephadex A25 were also shown by van Wezel to support cell growth.
The system proposed by van Wezel combines multiple surfaces with moveable surfaces and has the potential for innovative cellular manipulations and offers advantages in scale-up and environmental controls. Despite this potential, however, these suggested techniques have not been significantly exploited which is probably because of difficulties encountered in cell production due to deleterious effects which the beads seem to exhibit and which prevent good cell growth. For example, van Wezel's published data indicate that, even with bead washings and pretreatments, up to 75% of the inoculum has been lost using the DEAE-treated polydextran beads.